*lysine tRNA CUU (tRK1) encoded by nuclear genome and delivered to mitochondrial matrix [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17560369&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum described almost exclusively in the work of Ivan Tarassov]. Other lysine tRNAs are nuclear encoded-tRK2 which decodes UUU as lysine in the cytoplasm and mitochondrially encoded-tRK3 which decodes UUU for lysine in the mitochondria.

*lysine tRNA CUU (tRK1) encoded by nuclear genome and delivered to mitochondrial matrix [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17560369&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum described almost exclusively in the work of Ivan Tarassov]. Other lysine tRNAs are nuclear encoded-tRK2 which decodes UUU as lysine in the cytoplasm and mitochondrially encoded-tRK3 which decodes UUU for lysine in the mitochondria.

Gen'l info about RNases families

From Current Opinion in Structural Biology 2007 17:77 by James M Berger and Christoph W Müller "A particularly interesting family of ribonucleases that specifically cleave double-stranded RNA serves as the topic of the review by MacRae and Doudna. The RNase III group of RNA-processing enzymes currently attracts broad attention, because two family members, Dicer and Drosha, are responsible for processing RNA transcripts into microRNA (miRNAs) and short interfering RNAs (siRNAs). RNase III proteins are often multifunctional or multisubunit assemblies, and can be classified based on domain composition. Class I RNase III enzymes function as dimers, in which the RNase domains also act as dimerization domains, whereas class II and III family members are monomeric, forming a functional RNase from the internal fusion of two class I RNase III monomers. Comparing RNase III enzymes across a wide range of species leads the authors to conclude that RNase III enzymes use accessory domains as determinants of substrate specificity. For Dicer and Drosha, these accessory domains are the PAZ domain and the additional DGCR8 protein, respectively. Substrate specificity and catalytic domains are spatially separated and, in some instances, it appears that the RNase can precisely measure the distance between the RNA recognition and cleavage sites by using an internal scaffold element that functions as a molecular ruler. Given the number of different types of small RNAs and their importance in gene regulation and other cellular processes, there are sure to be many fundamental insights that will arise from the continued study of this essential protein family."

Part 2: Guide RNAs

Structural requirements for guide RNAs

Cell components needed for moving guide RNA to mt

piggy back on existing import system

lysine tRNA CUU (tRK1) encoded by nuclear genome and delivered to mitochondrial matrix described almost exclusively in the work of Ivan Tarassov. Other lysine tRNAs are nuclear encoded-tRK2 which decodes UUU as lysine in the cytoplasm and mitochondrially encoded-tRK3 which decodes UUU for lysine in the mitochondria.

Experimental checkpoints for regulated expression

Part 1: Rnt1p in mt

regulatable promoter driving second copy of Rnt1p for mt.

might use GalS promoter which is undetectable in glucose, leaky in YPEG and induced in Gal. Alternatively might want doxycyline, with minimal physiological consequence to regulation by tet. "off" may not be as tight so will need to check uninduced/induced by Northern.